GEL ELECTROPHORESIS

Exercise 5

Announcements

Post Lab 5 is due before your lab meets next week. Pre Lab 8 will be made available the week of March

17 and due before your Lab 8. LNA DNA/DNA Electrophoresis is assigned today

and due next week before Exercise 6 (Exam 1 Review).

Sign up for Exam I. Exam I will take place during your lab period the Week of March 17.

There will be a Review Sheet for Exam I posted under Week 7 by March 3.

Exam I covers material from Exercises 1-5. The last day to drop MCB 151 is March 14.

Week 5 Itinerary

Find your samples from week 4 and store them on ice

Wear gloves (EtBr contamination) Check out kits Pick up gel Load and run gel Take photos of gel (make 3 copies so you each

have one for your notebook assignment) Make sure you understand your photo before

leaving

CAUTION

Dispose of ALL waste properly Soft EtBr contaminated waste goes in biohazard bags Gel waste goes in the plastic jug located next to the

transilluminator UV Light

UV light will damage your eyes Ethidium Bromide (EtBr)

Present in the agarose, gel rigs, benches, etc. Don’t touch your face or hair while handling. Take care not to get EtBr on your lab manual or

notebook

DNA Gel Electrophoresis

Negatively charged DNA is pulled toward the anode

Large pieces/fragments of DNA do not move as fast as smaller ones

+

-C B BH H

+

-C B BH H

+

-C B BH H

+

-C B BH H

+

-C B BH H

+

-C B BH H

We can convert migration distances into fragment size (kB) by using a standard curve (Size versus migration distance)

+

- C B BH Hλ

6.5 kB5.0 kB4.0 kB3.5 kB3.0 kB

2.0 kB

1.0 kB

0.5 kB

Known

Measure

+

- C B BH Hλ

6.5 kB5.0 kB4.0 kB3.5 kB3.0 kB

2.0 kB

1.0 kB

0.5 kB

Known

Standard kbases

Distancemm

6.5 4

5.0 6

4.0 8

3.5 9

3.0 10

2.0 13

1.0 19

0.5 24

B (mm)

BH (mm)

H (mm)

9 8 9

24

Distance Migrated (mm)

4 6 8 10 12 14 16 18 20 22 24

0.1

0.2

0.3

0.5

1.0

2.0

3.0

5.0

10.0

Size (kBases)

6.5 kB5.0 kB4.0 kB3.5 kB3.0 kB

2.0 kB

1.0 kB

0.5 kB

Known

4 mm

6 mm8 mm9 mm10 mm

13 mm

19 mm

24 mm

Measured

Standard CurveSiz

e (

Kb)

Distance Migrated (mm)

4 6 8 10 12 14 16 18 20 22 24

0.1

0.2

0.3

0.5

1.0

2.0

3.0

5.0

10.0

Size (kBases)

6.5 kB5.0 kB4.0 kB3.5 kB3.0 kB

2.0 kB

1.0 kB

0.5 kB

Known

4 mm

6 mm8 mm9 mm10 mm

13 mm

19 mm

24 mm

Measured

Siz

e (

Kb)

B BH Hmm mm mm9 98

24

B BH Hmm mm mm

H

B

Plasmid Size = ? kBases

You need to make a “map” like this for your plasmid.

•What gel do you expect? (B, H, BH)

•Be sure to include plasmid size on your map.

Size of this fragment?

Size of this fragment?

Trouble Shooting (when gel bands are less than ideal)

Consider: Different amounts of supercoiling Partial Digests Small fragments are harder to see (less

DNA so less EtBr incalating)